PROJECT SUMMARY Fusobacterium nucleatum is a key colonizer in the development of oral biofilms or dental plaque by virtue of its interaction, termed coaggregation, with early and late colonizers of the oral biofilms. This oral pathogen is also associated with adverse pregnancy outcomes and colorectal cancer. However, little is known about the mechanisms of fusobacterial pathogenesis and associated factors; to date, only three coaggregation factors including the outer membrane adhesin RadD have been reported. This is mainly due to the lack of robust genetic tools for this organism. To overcome this bottleneck in fusobacterial research, we have recently developed a facile allelic exchange method and adopted a Tn5-based transposon system for F. nucleatum to generate a large library of Tn5 mutants with more than 10-fold genome coverage. In a pilot study, we screened for mutants that are defective in coaggregation with the early colonizer Streptococcus oralis and found several novel factors, in addition to RadD, which are required for fusobacterial coaggregation. We also revealed that genetic disruption of a novel two-component system (TCS) named CarRS (car for co-aggregation regulating factor) altered radD expression. Altogether, we hypothesize that fusobacterial coaggregation is a multifactorial process that is modulated by the TCS CarRS in response to the environmental signals in the oral cavity. To test this hypothesis, we aim to elucidate the molecular mechanism of CarRS-modulated coaggregation via gene regulation. Secondly, by generating a two-allele library of well-defined Tn5 mutants, we aim to reveal all nonessential factors that are involved in fusobacterial coaggregation with the periodontal pathogens Porphyromonas gingivalis and Streptococcus mutans, as well as the early colonizers S. oralis and Actinomyces oris. The studies presented here will reveal for the first time not only the regulation mechanism of CarSR-mediated coaggregation, but also a comprehensive view of fusobacterial factors required for polymicrobial interactions. Equally important is our generation of the first well-defined mutant library that provides a valuable tool to other investigators to examine many important processes involving fusobacterial pathogenesis. Our studies will also yield clues to the mechanisms of dental biofilm formation and may lead to the development of novel agents that control dental biofilm formation.